Project Summary/Abstract: To orchestrate neurotransmission, more than one thousand proteins are present at the presynaptic terminal and which either directly or indirectly interact with the synaptic vesicle. This high degree of convergence of presynaptic functions onto the synaptic vesicle has led to a ?vesicocentric? view of neurotransmission that focuses on the synaptic vesicle as the central organelle in synaptic function. As a result, much effort in molecular neurobiology in the past decades has been spent to identify the proteins present on synaptic vesicles and how they function to control neurotransmitter release. In addition, synaptic vesicles are also used as a model trafficking organelle to understand the mechanism utilized by the eukaryotic cell to carry out membrane fusion and trafficking. Because of these central roles synaptic vesicles play in neurotransmission as well as a model system for understanding membrane trafficking in general, it is critically important to develop a detailed and quantitative understanding of the organization of the synaptic vesicle. Synaptic vesicle is the smallest organelle present in the cell. From electron-microscopy (EM) measurements, synaptic vesicle has a diameter of ~40nm. In comparison, a ribosome observed under EM has a ?diameter? of 20-30nm depending on the contrast employed. In terms of size, therefore, synaptic vesicles are similar in many ways to a very large macromolecular complex, and as such, amenable to high- resolution single-molecule imaging and quantitative biophysical studies. Synaptic vesicle, however, is too large and disorganized to be studied with established structural methods, such as crystallography or cryoEM. The goal of the proposed project is to go beyond the compositional studies conducted in the past decade so as to unravel how proteins are spatially organized and interacting on the synaptic vesicle, and to construct a molecular/structural view of the synaptic vesicle. To achieve this, we have the following Aims: Aim 1: Single-Molecule Positional Mapping and Counting of Membrane Proteins on Synaptic Vesicles with Photoswitchable Pdots - this study will tell us which proteins are always located at the same positions on the vesicle, which proteins are adjacent to each other, and which are randomly distributed on the vesicle. Aim 2: Single-Molecule Orientation Mapping of Membrane Proteins on Synaptic Vesicles with Polarized Pdots - we know proteins are extremely crowded on the vesicle, but if proteins are interacting with each other or form a complex, then they are not only in close proximity but also would have the same rotational rate on the membrane or have very limited rotational freedom. This experiment will inform us which proteins are forming a complex and also whether they are in the same complex. Aim 3: Positional and Orientational Mapping of Membrane Proteins on Different Types of Synaptic Vesicle Enabled by a Nanoscale Sorter - different types of SVs might arrange the membrane proteins differently. We will use a nanoscale sorter to isolate and study different types of synaptic vesicles.